RESUMEN
KEY POINTS: Tymothy syndrome (TS) is a multisystem disorder featuring cardiac arrhythmias, autism and adrenal gland dysfunction that originates from a de novo point mutation in the gene encoding the Cav1.2 (CACNA1C) L-type channel. To study the role of Cav1.2 channel signals in autism, the autistic TS2-neo mouse has been generated bearing the G406R point-mutation associated with TS type-2. Using heterozygous TS2-neo mice, we report that the G406R mutation reduces the rate of inactivation and shifts leftward the activation and inactivation of L-type channels, causing marked increase of resting Ca2+ influx ('window' Ca2+ current). The increased 'window current' causes marked reduction of NaV channel density, switches normal tonic firing to abnormal burst firing, reduces mitochondrial metabolism, induces cell swelling and decreases catecholamine release. Overnight incubations with nifedipine rescue NaV channel density, normal firing and the quantity of catecholamine released. We provide evidence that chromaffin cell malfunction derives from altered Cav1.2 channel gating. ABSTRACT: L-type voltage-gated calcium (Cav1) channels have a key role in long-term synaptic plasticity, sensory transduction, muscle contraction and hormone release. A point mutation in the gene encoding Cav1.2 (CACNA1C) causes Tymothy syndrome (TS), a multisystem disorder featuring cardiac arrhythmias, autism spectrum disorder (ASD) and adrenal gland dysfunction. In the more severe type-2 form (TS2), the missense mutation G406R is on exon 8 coding for the IS6-helix of the Cav1.2 channel. The mutation causes reduced inactivation and induces autism. How this occurs and how Cav1.2 gating-changes alter cell excitability, neuronal firing and hormone release on a molecular basis is still largely unknown. Here, using the TS2-neo mouse model of TS we show that the G406R mutation altered excitability and reduced secretory activity in adrenal chromaffin cells (CCs). Specifically, the TS2 mutation reduced the rate of voltage-dependent inactivation and shifted leftward the activation and steady-state inactivation of L-type channels. This markedly increased the resting 'window' Ca2+ current that caused an increased percentage of CCs undergoing abnormal action potential (AP) burst firing, cell swelling, reduced mitochondrial metabolism and decreased catecholamine release. The increased 'window' Ca2+ current caused also decreased NaV channel density and increased steady-state inactivation, which contributed to the increased abnormal burst firing. Overnight incubation with the L-type channel blocker nifedipine rescued the normal AP firing of CCs, the density of functioning NaV channels and their steady-state inactivation. We provide evidence that CC malfunction derives from the altered Cav1.2 channel gating and that dihydropyridines are potential therapeutics for ASD.
Asunto(s)
Potenciales de Acción , Trastorno Autístico/genética , Canales de Calcio Tipo L/genética , Células Cromafines/metabolismo , Exocitosis , Síndrome de QT Prolongado/genética , Sindactilia/genética , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/metabolismo , Catecolaminas/metabolismo , Células Cultivadas , Células Cromafines/efectos de los fármacos , Células Cromafines/fisiología , Activación del Canal Iónico , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Nifedipino/farmacología , Mutación Puntual , Canales de Sodio/metabolismoRESUMEN
miR-21 is overexpressed in tumors and it displays oncogenic activity. Here, we show that expression of miR-21 in primary tumors anticorrelates with KRIT1/CCM1, an interacting partner of the Ras-like GTPase Rap1, involved in Cerebral Cavernous Malformations (CCM). We present evidences that miR-21 silences KRIT1 by targeting its mRNA 3'UTR and that this interaction is involved in tumor growth control. In fact, miR-21 over-expression or KRIT1 knock-down promote anchorage independent tumor cell growth compared to controls, whereas the opposite is observed when anti-miR-21 or KRIT1 overexpression are employed. Our findings suggest that miR-21 promotes tumor cell growth, at least in part, by down-modulating the potential tumor suppressor KRIT1.
Asunto(s)
Neoplasias de la Mama/patología , Neoplasias de la Mama/fisiopatología , Regulación Neoplásica de la Expresión Génica/genética , MicroARNs/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Línea Celular Tumoral , Proliferación Celular , Humanos , Proteína KRIT1 , Proteínas Asociadas a Microtúbulos/genética , Proteínas Proto-Oncogénicas/genéticaRESUMEN
KRIT1 is a disease gene responsible for Cerebral Cavernous Malformations (CCM). It encodes for a protein containing distinct protein-protein interaction domains, including three NPXY/F motifs and a FERM domain. Previously, we isolated KRIT1B, an isoform characterized by the alternative splicing of the 15th coding exon and suspected to cause CCM when abnormally expressed. Combining homology modeling and docking methods of protein-structure and ligand binding prediction with the yeast two-hybrid assay of in vivo protein-protein interaction and cellular biology analyses we identified both structural and functional differences between KRIT1A and KRIT1B isoforms. We found that the 15th exon encodes for the distal beta-sheet of the F3/PTB-like subdomain of KRIT1A FERM domain, demonstrating that KRIT1B is devoid of a functional PTB binding pocket. As major functional consequence, KRIT1B is unable to bind Rap1A, while the FERM domain of KRIT1A is even sufficient for this function. Furthermore, we found that a functional PTB subdomain enables the nucleocytoplasmic shuttling of KRIT1A, while its alteration confers a restricted cytoplasmic localization and a dominant negative role to KRIT1B. Importantly, we also demonstrated that KRIT1A, but not KRIT1B, may adopt a closed conformation through an intramolecular interaction involving the third NPXY/F motif at the N-terminus and the PTB subdomain of the FERM domain, and proposed a mechanism whereby an open/closed conformation switch regulates KRIT1A nuclear translocation and interaction with Rap1A in a mutually exclusive manner. As most mutations found in CCM patients affect the KRIT1 FERM domain, the new insights into the structure-function relationship of this domain may constitute a useful framework for understanding molecular mechanisms underlying CCM pathogenesis.
Asunto(s)
Malformaciones Vasculares del Sistema Nervioso Central/fisiopatología , Hemangioma Cavernoso del Sistema Nervioso Central/fisiopatología , Proteínas Asociadas a Microtúbulos/fisiología , Dominios y Motivos de Interacción de Proteínas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Animales , Células COS , Línea Celular , Malformaciones Vasculares del Sistema Nervioso Central/genética , Chlorocebus aethiops , Simulación por Computador , Células HeLa , Hemangioma Cavernoso del Sistema Nervioso Central/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína KRIT1 , Ratones , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Modelos Moleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Mutación Puntual , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Estructura Secundaria de Proteína , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Técnicas del Sistema de Dos Híbridos , Proteínas de Unión al GTP rap1/química , Proteínas de Unión al GTP rap1/genética , Proteínas de Unión al GTP rap1/metabolismoRESUMEN
Myeloid-derived suppressor cells (MDSC) include immature monocytic (M-MDSC) and granulocytic (PMN-MDSC) cells that share the ability to suppress adaptive immunity and to hinder the effectiveness of anticancer treatments. Of note, in response to IFNγ, M-MDSCs release the tumor-promoting and immunosuppressive molecule nitric oxide (NO), whereas macrophages largely express antitumor properties. Investigating these opposing activities, we found that tumor-derived prostaglandin E2 (PGE2) induces nuclear accumulation of p50 NF-κB in M-MDSCs, diverting their response to IFNγ toward NO-mediated immunosuppression and reducing TNFα expression. At the genome level, p50 NF-κB promoted binding of STAT1 to regulatory regions of selected IFNγ-dependent genes, including inducible nitric oxide synthase (Nos2). In agreement, ablation of p50 as well as pharmacologic inhibition of either the PGE2 receptor EP2 or NO production reprogrammed M-MDSCs toward a NOS2low/TNFαhigh phenotype, restoring the in vivo antitumor activity of IFNγ. Our results indicate that inhibition of the PGE2/p50/NO axis prevents MDSC-suppressive functions and restores the efficacy of anticancer immunotherapy. SIGNIFICANCE: Tumor-derived PGE2-mediated induction of nuclear p50 NF-κB epigenetically reprograms the response of monocytic cells to IFNγ toward an immunosuppressive phenotype, thus retrieving the anticancer properties of IFNγ. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/13/2874/F1.large.jpg.
Asunto(s)
Diferenciación Celular , Neoplasias Colorrectales/patología , Dinoprostona/farmacología , Monocitos/patología , Células Supresoras de Origen Mieloide/patología , Subunidad p50 de NF-kappa B/metabolismo , Neoplasias Pancreáticas/patología , Animales , Apoptosis , Proliferación Celular , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/metabolismo , Humanos , Tolerancia Inmunológica , Interferón gamma/metabolismo , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/inmunología , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/metabolismo , Células Supresoras de Origen Mieloide/efectos de los fármacos , Células Supresoras de Origen Mieloide/inmunología , Células Supresoras de Origen Mieloide/metabolismo , Subunidad p50 de NF-kappa B/genética , Óxido Nítrico/metabolismo , Oxitócicos/farmacología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/metabolismo , Células Tumorales CultivadasRESUMEN
Using two-hybrid screening, we isolated the integrin cytoplasmic domain-associated protein (ICAP-1), an interactor for the COOH terminal region of the beta1A integrin cytoplasmic domain. To investigate the role of ICAP-1 in integrin-mediated adhesive function, we expressed the full-length molecule in NIH3T3 cells. ICAP-1 expression strongly prevents NIH3T3 cell spreading on extracellular matrix. This inhibition is transient and can be counteracted by coexpression of a constitutively activated mutant of Cdc42, suggesting that ICAP-1 acts upstream of this GTPase. In addition, we found that ICAP-1 binds both to Cdc42 and Rac1 in vitro, and its expression markedly inhibits activation of these GTPases during integrin-mediated cell adhesion to fibronectin as detected by PAK binding assay. In the attempt to define the molecular mechanism of this inhibition, we show that ICAP-1 reduces both the intrinsic and the exchange factor-induced dissociation of GDP from Cdc42; moreover, purified ICAP-1 displaces this GTPase from cellular membranes. Together, these data show for the first time that ICAP-1 regulates Rho family GTPases during integrin-mediated cell matrix adhesion, acting as guanine dissociation inhibitor.
Asunto(s)
Proteínas Portadoras/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Proteínas de Unión al GTP rho/metabolismo , Células 3T3 , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Células COS , División Celular , Tamaño de la Célula , Guanosina Difosfato/metabolismo , Integrinas/metabolismo , Ratones , Datos de Secuencia Molecular , Unión Proteica , Ratas , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Especificidad por Sustrato , Factores de Tiempo , Técnicas del Sistema de Dos Híbridos , Proteína de Unión al GTP cdc42/antagonistas & inhibidores , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/antagonistas & inhibidores , Proteína de Unión al GTP rac1/metabolismo , Proteínas de Unión al GTP rho/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Although tumor-associated macrophages (TAM) display a M2-skewed tumor-promoting phenotype in most cancers, in colorectal cancer, both TAM polarization and its impact remain controversial. We investigated the role of the M2-polarizing p50 NF-κB subunit in orchestrating TAM phenotype, tumor microenvironment composition, and colorectal cancer progression. We first demonstrated, by parallel studies in colitis-associated cancer (CAC) and in genetically driven ApcMin mouse models, that the p50-dependent inhibition of M1-polarized gut inflammation supported colorectal cancer development. In accordance with these studies, p50-/- mice displayed exacerbated CAC with fewer and smaller tumors, along with enhanced levels of M1/Th1 cytokines/chemokines, including IL12 and CXCL10, whose administration restrained CAC development in vivo The inflammatory profile supporting tumor resistance in colons from p50-/- tumor bearers correlated inversely with TAM load and positively with both recruitment of NK, NKT, CD8+ T cells and number of apoptotic tumor cells. In agreement, myeloid-specific ablation of p50 promoted tumor resistance in mice, whereas in colorectal cancer patients, a high number of p50+ TAMs at the invasive margin was associated with decreased IL12A and TBX21 expression and worse postsurgical outcome. Our findings point to p50 involvement in colorectal cancer development, through its engagement in the protumor activation of macrophages, and identify a candidate for prognostic and target therapeutic intervention. Cancer Immunol Res; 6(5); 578-93. ©2018 AACR.
Asunto(s)
Neoplasias Colorrectales/patología , Mediadores de Inflamación/fisiología , Inflamación/complicaciones , Subunidad p50 de NF-kappa B/fisiología , Animales , Polaridad Celular/genética , Células Cultivadas , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/metabolismo , Progresión de la Enfermedad , Humanos , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Mediadores de Inflamación/metabolismo , Activación de Macrófagos/genética , Macrófagos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Subunidad p50 de NF-kappa B/genética , Subunidad p50 de NF-kappa B/metabolismo , Microambiente Tumoral/genética , Microambiente Tumoral/inmunologíaRESUMEN
The coordinate modulation of the cellular functions of cadherins and integrins plays an essential role in fundamental physiological and pathological processes, including morphogenesis, tissue differentiation and renewal, wound healing, immune surveillance, inflammatory response, tumour progression, and metastasis. However, the molecular mechanisms underlying the fine-balanced relationship between cadherin and integrin functions are still elusive. This review focuses on recent findings on the involvement of the small GTPase Rap1 in the regulation of cadherin- and integrin-dependent cell adhesion and signal transduction. In particular, it highlights some of the novel results recently obtained that raise the possibility of a pivotal role for Rap1 in the functional crosstalk between cadherins and integrins, suggesting interesting new regulatory mechanisms.
Asunto(s)
Cadherinas/metabolismo , Integrinas/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rap1/metabolismo , Animales , Adhesión Celular , Endosomas/metabolismo , Humanos , Modelos Biológicos , Transporte de ProteínasRESUMEN
Cerebral cavernous malformations (CCM) are vascular malformations, mostly located in the central nervous system, which occur in 0.1-0.5% of the population. They are characterized by abnormally enlarged and often leaking capillary cavities without intervening neural parenchyma. Some are clinically silent, whereas others cause seizures, intracerebral haemorrhage or focal neurological deficits. These vascular malformations can arise sporadically or may be inherited as an autosomal dominant condition with incomplete penetrance. At least 45% of families affected with cerebral cavernous malformations harbour a mutation in Krev interaction trapped-1 (Krit1) gene (cerebral cavernous malformation gene-1, CCM1). This gene contains 16 coding exons which encode a 736-amino acid protein containing three ankyrin repeats and a FERM domain. Neither the CCM1 pathogenetic mechanisms nor the function of the Krit1 protein are understood so far, although several hypotheses have been inferred from the predicted consequences of Krit1 mutations as well as from the identification of Krit1 as a binding partner of Rap1A, ICAP1A and microtubules. Here, we report the identification of Krit1B, a novel Krit1 isoform characterized by the alternative splicing of the 15th coding exon. We show that the Krit1B splice isoform is widely expressed in mouse cell lines and tissues, whereas its expression is highly restricted in human. In addition, we developed a real-time PCR strategy to accurately quantify the relative ratio of the two Krit1 alternative transcripts in different tissues, demonstrating a Krit1B/Krit1A ratio up to 20% in mouse thymus, but significantly lower ratios in other tissues. Bioinformatic analysis using exon/gene-prediction, comparative alignment and structure analysis programs supported the existence of Krit1 alternative transcripts lacking the 15th coding exon and showed that the splicing out of this exon occurs outside of potentially important Krit1 structural domains but in a region required for association with Rap1A, suggesting a subtle, yet important effect on the protein function. Our results indicate that maintenance of a proper ratio between Krit1A and Krit1B could be functionally relevant and suggest that the novel Krit1B isoform might expand our understanding of the role of Krit1 in CCM1 pathogenesis.
Asunto(s)
Empalme Alternativo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Proto-Oncogénicas/genética , Animales , Repetición de Anquirina/genética , Secuencia de Bases , Células COS , Línea Celular , Línea Celular Tumoral , Chlorocebus aethiops , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Bases de Datos de Ácidos Nucleicos , Expresión Génica , Humanos , Proteína KRIT1 , Ratones , Datos de Secuencia Molecular , Células 3T3 NIH , Isoformas de Proteínas/genética , Análisis de Secuencia de ADNRESUMEN
KRIT1 is a gene responsible for Cerebral Cavernous Malformations (CCM), a major cerebrovascular disease characterized by abnormally enlarged and leaky capillaries that predispose to seizures, focal neurological deficits, and fatal intracerebral hemorrhage. Comprehensive analysis of the KRIT1 gene in CCM patients has suggested that KRIT1 functions need to be severely impaired for pathogenesis. However, the molecular and cellular functions of KRIT1 as well as CCM pathogenesis mechanisms are still research challenges. We found that KRIT1 plays an important role in molecular mechanisms involved in the maintenance of the intracellular Reactive Oxygen Species (ROS) homeostasis to prevent oxidative cellular damage. In particular, we demonstrate that KRIT1 loss/down-regulation is associated with a significant increase in intracellular ROS levels. Conversely, ROS levels in KRIT1(-/-) cells are significantly and dose-dependently reduced after restoration of KRIT1 expression. Moreover, we show that the modulation of intracellular ROS levels by KRIT1 loss/restoration is strictly correlated with the modulation of the expression of the antioxidant protein SOD2 as well as of the transcriptional factor FoxO1, a master regulator of cell responses to oxidative stress and a modulator of SOD2 levels. Furthermore, we show that the KRIT1-dependent maintenance of low ROS levels facilitates the downregulation of cyclin D1 expression required for cell transition from proliferative growth to quiescence. Finally, we demonstrate that the enhanced ROS levels in KRIT1(-/-) cells are associated with an increased cell susceptibility to oxidative DNA damage and a marked induction of the DNA damage sensor and repair gene Gadd45alpha, as well as with a decline of mitochondrial energy metabolism. Taken together, our results point to a new model where KRIT1 limits the accumulation of intracellular oxidants and prevents oxidative stress-mediated cellular dysfunction and DNA damage by enhancing the cell capacity to scavenge intracellular ROS through an antioxidant pathway involving FoxO1 and SOD2, thus providing novel and useful insights into the understanding of KRIT1 molecular and cellular functions.
Asunto(s)
Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Western Blotting , Línea Celular , Proliferación Celular , Células Cultivadas , Femenino , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Humanos , Inmunoprecipitación , Proteína KRIT1 , Masculino , Ratones , Proteínas Asociadas a Microtúbulos/genética , Reacción en Cadena de la Polimerasa , Embarazo , Proteínas Proto-Oncogénicas/genética , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismoRESUMEN
The coordinate modulation of cadherin and integrin functions plays an essential role in fundamental physiological and pathological processes, including morphogenesis and cancer. However, the molecular mechanisms underlying the functional crosstalk between cadherins and integrins are still elusive. Here, we demonstrate that the small GTPase Rap1, a crucial regulator of the inside-out activation of integrins, is a target for E-cadherin-mediated outside-in signaling. In particular, we show that a strong activation of Rap1 occurs upon adherens junction disassembly that is triggered by E-cadherin internalization and trafficking along the endocytic pathway. By contrast, Rap1 activity is not influenced by integrin outside-in signaling. Furthermore, we demonstrate that the E-cadherin endocytosis-dependent activation of Rap1 is associated with and controlled by an increased Src kinase activity, and is paralleled by the colocalization of Rap1 and E-cadherin at the perinuclear Rab11-positive recycling endosome compartment, and the association of Rap1 with a subset of E-cadherin-catenin complexes that does not contain p120ctn. Conversely, Rap1 activity is suppressed by the formation of E-cadherin-dependent cell-cell junctions as well as by agents that inhibit either Src activity or E-cadherin internalization and intracellular trafficking. Finally, we demonstrate that the E-cadherin endocytosis-dependent activation of Rap1 is associated with and is required for the formation of integrin-based focal adhesions. Our findings provide the first evidence of an E-cadherin-modulated endosomal signaling pathway involving Rap1, and suggest that cadherins may have a novel modulatory role in integrin adhesive functions by fine-tuning Rap1 activation.